The techniques of modern biotechnology have made possible the identification of the genetic elements, or genes, which control the characteristics of living organisms. The principle manner by which most genes cause effects in organisms is by encoding the construction of proteins. Thus in studying genes, it is often desired to produce a protein from the protein coding DNA from the gene, to study what the protein is, or what the protein does, or to perform some useful reaction with the protein. Sometimes a protein is expressed by inserting the entire gene, or an artificial construct carrying the protein coding sequence in an expression vector, into a suitable host cell so that the host cells can be grown to produce the protein. Another technique is to produce the protein in vitro directly from a gene or an artificial genetic construct in a cell free protein synthesis process. In vitro techniques for protein synthesis have the advantage that the protein can be produced directly from the encoding DNA without the necessity for intermediate culture and proliferation of transformed cells. In vitro protein synthesis provides the further advantage of allowing the production of proteins that are typically difficult or impossible to express in living cells, such as toxins or proteins containing amino acids that do not normally occur in living cells.
Methods for in vitro transcription and translation of DNA to produce protein have been known for many years. The earliest documented descriptions of in vitro protein synthesis were developed in prokaryotic systems that utilized bacterial transcriptional and translational components to produce proteins in a coupled reaction. A common prokaryotic system, known as an E. coli S-30 cell free extract, was first described in a systematic way by Zubay, Annual Review of Genetics 7:267–287 (1973). Others have written articles and reviews on how to better make and use such S-30 extracts. In addition, kits for the in vitro production of proteins from DNA have been commercialized based on the use of S-30 extracts. Such kits are sold by several manufacturers. More recently, systems have been developed for transcription and translation using eukaryotic cell free extracts, particularly those based on the use of rabbit reticulocyte lysate or wheat germ extract. U.S. Pat. Nos. 5,324,637 and 5,895,753 describe systems for in vitro transcription and translation of protein.
Both prokaryotic and eukaryotic cell free extracts for transcription and translation are sold today in commerce. In general, the researcher using an in vitro transcription and translation system wants the process to produce an optimum amount of the full length target protein and wants to minimize the amount of non-targeted protein and/or less than full length protein which is made. While prokaryotic systems are inherently simpler to use, the eukaryotic systems are thought to be superior for some applications. In particular, E. coli S-30 extracts are convenient to make and use but tend to produce a greater percentage of non-fill length protein than eukaryotic extracts. The production of unwanted protein or polypeptide products is generally observed by the presence of a variety of proteins different in size than the full-length target protein when the reaction products are visualized by gel electrophoresis. The non-full length proteins are thought to arise from several sources, which fall into two primary categories.
First, a certain amount of background non-target proteins is produced in S-30 systems due to transcription from other E. coli promoters present in the extract, either from residual E. coli genomic DNA left in the extract or from other promoters on plasmids or other vectors which carry the target gene. For example, most plasmids carry a gene which encodes a protein, such as β-lactamase, that confers resistance to a selective drug, such as ampicillin, in addition to the target gene. When such a plasmid is used as the template for in vitro protein synthesis in an E. coli S-30 extract, both the target gene and the drug resistance gene are transcribed by the RNA polymerase and protein is therefore produced from both genes. A minor amount of non-target background may also be derived from non-specific initiation of transcription of E. coli RNA polymerase on non-promoter sites on DNA present in the extract. This phenomenon is known to occur under some conditions but is unlikely to be significant under the typical reaction conditions for in vitro protein synthesis. Background can also be derived from translation of residual E. coli mRNA present in the extract. Some of the strategies that have been used to minimize the contribution of endogenous DNA and mRNA to non-target background include a pre-incubation step to allow run-off of ribosomes engaged in translation of endogenous mRNA and treatment with Ca2+ dependent nuclease to preferentially degrade endogenous DNA and mRNA. However, it has not generally been possible to entirely eliminate or remove non-target synthesis with these steps, since some DNA and mRNA typically remains after the treatment, and extended incubation or over-treatment with nuclease results in unacceptably low levels of protein synthesis activity, presumably due to damage to other species of RNA, including rRNA and tRNA that participate in protein synthesis. A different approach has been taken to direct the exclusive synthesis of target proteins by using T7 RNA polymerase for transcription in E. coli extracts in the presence of rifampicin to inhibit E. coli RNA polymerase (Nevins and Pratt, FEBS Lett 291:259–263(1991)).
The second major category of background in E. coli extracts results in the generation of smaller, truncated forms of the target protein. These forms arise from one or more causes, including: (1) initiation of protein synthesis at internal AUG start codons other than the authentic N-terminal AUG, (2) synthesis of incomplete polypeptide chains due to premature termination of translation, (3) degradation of template DNA and/or RNA transcripts by nucleases present in the extract, and (4) degradation of the target protein by proteases present in the extract. The degradation of linear DNA templates has been approached by using extracts derived from strains deficient in one or more enzymes of the RecBCD complex (Yang et al., Proc. Natl. Acad. Sci. USA 77:7029–7033 (1980)). Strains deficient in ompT and lon proteases have also be used to minimize proteolytic degradation (Kohrer et al., Eur. J. Biochem. 236:234–239 (1996)). While these examples appear to alleviate some degradation activity, there are many additional activities in cells that have not been possible to eliminate due to their being essential for cellular viability. In addition, there is not a method currently known in the art that generally addresses internal initiation or premature termination, which are believed to be significant causes of non-full length background. One may imagine that the use of stains deficient in one or more of the major ribonuclease activities present in E. coli may produce extracts exhibiting greater synthesis of full length proteins, but there have been no reports of success using this approach. The various RNA degradation pathways in E. coli and the interactions of enzymes and other proteins involved therein, both in vivo and in vitro, are still being elucidated.
An important contributor to the degradation of RNA transcripts in cells appears to be the RNA degradosome, a multi-protein complex that is involved in RNA turnover and metabolism. The degradosome is organized around the enzyme RNase E which contains binding sites for other key protein components of the complex. See, for example, Lopez et al., Mol. Micro. 33(1). 189–199 (1999) and Vanzo et al., Genes & Develop. 12:2770–2781 (1998). What, if any, role the RNA degradosome or RNase E plays in E coli transcription and translation extracts was heretofore unknown.
The cell free S-30 system, as devised by Zubay and modified by others, traditionally involves the preparation of an extract and a supplemental mix. The extract contains all the enzymes and factors from the E. coli necessary for transcription and translation. The supplemental mix includes nucleotide triphosphates, tRNA, amino acids and an energy regenerating system plus certain co-factors and salts and ions. The making and using of such extracts and supplemental mixes has been documented for more than 30 years and have been sold as kits by commercial companies for some time. While such extracts are conventionally made using E. coli, there is no technical reason why such extracts cannot be made from any number of possible prokaryotic hosts.
A typical S-30 extract is made by first culturing the E. coli cells and harvesting them. The E. coli cells are then lysed or broken with a French pressure cell or other cell disruption device. The resulting lysate is then centrifuged to remove the cellular debris and other solid matter and the supernatant is saved for further processing. The supernatant is then combined with a pre-incubation buffer and incubated. Sometimes a microccocal nuclease treatment step is included to remove contaminating DNA and mRNA from the original host cells. The extract is then dialyzed and stored frozen until needed.
The typical supplemental mix that is added to an S-30 coupled transcription and translation reaction contains buffers, such as Tris-acetate, dithiothreitol (DTT), the NTPs (ATP, CTP, GTP, and UTP), phosphoenol pyruvate, pyruvate kinase, amino acids (typically 10 of the 20 naturally occurring amino acids, leaving one out to permit addition of a radio-labeled amino acid or analog), polyethylene glycol (PEG), folinic acid, cAMP, tRNA, ammonium acetate, potassium acetate, calcium acetate and an optimized concentration of magnesium acetate. These types of components, or their equivalents, are mixed together in a process separate from the production of the S-30 extract. Supplemental mix is also typically stored frozen for later use in S-30 coupled reactions.
In the prior art a typical reaction conducted to produce in vitro protein from DNA using a E. coli S-30 extract involves the following components mixed together in a microcentrifuge tube, typically in a total volume of 25 to 50 microliters: 1. S-30 extract; 2. supplemental mix; 3. one or more additional amino acids (unlabeled or labeled); 4. water; and 5. the DNA template. This combination reaction is incubated for some period of time, typically an hour at 30–37° C. and the quantity and quality of the in vitro synthesized proteins is examined by a variety of methods.